Photobody formation spatially segregates two opposing phytochrome B signaling actions of PIF5 degradation and stabilization

Abstract

Photoactivation of the plant photoreceptor and thermosensor phytochrome B (PHYB) triggers its condensation into subnuclear membraneless organelles named photobodies (PBs). However, the function of PBs in PHYB signaling remains frustratingly elusive. Here, we found that PHYB recruits PHYTOCHROME-INTERACTING FACTOR 5 (PIF5) to PBs. Surprisingly, PHYB exerts opposing roles in degrading and stabilizing PIF5. Perturbing PB size by overproducing PHYB provoked a biphasic PIF5 response: while a moderate increase in PHYB enhanced PIF5 degradation, further elevating the PHYB level stabilized PIF5 by retaining more of it in enlarged PBs. Conversely, reducing PB size by dim light, which enhanced PB dynamics and nucleoplasmic PHYB and PIF5, switched the balance towards PIF5 degradation. Together, these results reveal that PB formation spatially segregates two antagonistic PHYB signaling actions - PIF5 stabilization in PBs and PIF5 degradation in the surrounding nucleoplasm - which could enable an environmentally sensitive, counterbalancing mechanism to titrate nucleoplasmic PIF5 and environmental responses.

Fig. 1. Increasing PHYB abundance alters PB size and dynamics.

a Images of 4-d-old phyB-9, Col-0, gPBC-25, gPBC-29 and PBC seedlings grown under 10 μmol m⁻² s⁻¹ R light at 21 ^oC. b Hypocotyl length measurements of the seedlings shown in (a). Error bars represent the s.d. (n > 50 seedlings); the centers of the error bars indicate the mean. Different letters denote statistically significant differences in hypocotyl length (ANOVA, Tukey’s HSD, multiplicity adjusted p ≤ 0.05). c Immunoblots showing the PHYB levels in the seedlings described in (a). Actin was used as a loading control. The relative endogenous PHYB and PHYB-CFP levels normalized to the corresponding levels of actin are shown. The immunoblot experiments were independently repeated three times with similar results. d Confocal images showing representative PHYB-CFP PB patterns in cotyledon pavement epidermal cells from 4-d-old gPBC-25, gPBC-29 and PBC seedlings grown under 10 μmol m⁻² s⁻¹ R light at 21 ^oC. Scale bars are equal to 2 µm. e Quantification of the PB volume in cotyledon epidermal cells from gPBC-25, gPBC-29 and PBC as shown in (d). Different letters denote statistically significant differences in volume (ANOVA, Tukey’s HSD, multiplicity adjusted p < 0.05). f Quantification of the percentage of PHYB partitioned to PBs based on the total PHYB signal within the nuclei of gPBC-25, gPBC-29 and PBC as shown in (d). Box and whisker plots showing the percent of PHYB partitioned to PBs per nucleus. Different letters denote statistically significant differences in percentage (ANOVA, Tukey’s HSD, multiplicity adjusted p < 0.05). For (e, f), the numbers indicate the mean values. In the box and whisker plots, the boxes represent the 25th to 75th percentiles, and the bars are equal to the median. g Results of FRAP experiments showing normalized fluorescence recovery plotted over time for PHYB-CFP in PBs of pavement cell nuclei from 4-d-old gPBC-25 (black) and PBC (magenta) seedlings grown under 50 μmol m⁻² s⁻¹ R light. Values represent the mean, and error bars represent the s.e. of the mean of three biological replicates. The solid lines represent an exponential fit of the data. MF: mobile fraction; t_1/2 represents the half-time of fluorescence recovery. The source data underlying the hypocotyl measurements in (b), the immunoblots in (c), and the PB characterization in (e–g) are provided in the Source Data file.

Fig. 2. mCherry-PIF5 is a short-lived protein localized in PBs.

a Images of 4-d-old seedlings of Col-0, pif5-3 and two mCherry-PIF5/pif5-3 (#8 and #9) lines grown under 50 μmol m⁻² s⁻¹ R light at 16 ^oC or 27 ^oC. b Hypocotyl length measurements of the seedlings described in a. The open and gray bars represent hypocotyl length measurements at 16 ^oC and 27 ^oC, respectively. Error bars for the hypocotyl measurements represent the s.d. (n = 90 seedlings). Lowercase and uppercase letters denote statistically significant differences in hypocotyl lengths at 16 ^oC or 27 ^oC, respectively (ANOVA, Tukey’s HSD, multiplicity adjusted p < 0.05, n = 90 seedlings). The black number above the Col-0 data represents the percent increase in hypocotyl length at 27 ^oC compared to 16 ^oC (mean ± s.d., n = 90 seedlings). The pink bars show the relative response, defined as the hypocotyl response to 27 ^oC of a mutant or transgenic line relative to that of Col-0 (set at 100%). Error bars for the relative responses represent the s.e. of three biological replicates. The centers of all error bars indicate the mean. Pink numbers show the mean ± s.e. of the relative responses. Different pink letters denote statistically significant differences in the relative responses (ANOVA, Tukey’s HSD, multiplicity adjusted p < 0.05, n = 3 biological replicates). c Immunoblots showing the steady-state levels of PIF5 and mCherry-PIF5 in 4-d-old Col-0, pif5-3, and the mCherry-PIF5 lines (#8, #9) grown under 50 μmol m⁻² s⁻¹ R light at 16 ^oC or 27 ^oC. PIF5 and mCherry-PIF5 were detected using anti-PIF5 antibodies. Actin was used as a loading control. The relative levels of PIF5 or mCherry-PIF5 normalized to actin are shown. The asterisk indicates a nonspecific band. d Confocal images showing the colocalization of mCherry-PIF5 and PHYB in PBs in cotyledon pavement epidermal nuclei from 4-d-old mCherry-PIF5 line #8 seedlings grown under 50 μmol m⁻² s⁻¹ R light at 16 ^oC or 27 ^oC. PHYB (green) and Myc-tagged mCherry-PIF5 (red) were labeled via immunofluorescence staining using anti-PHYB and anti-Myc antibodies, respectively. Nuclei were stained with DAPI (blue). Scale bars are equal to 2 µm. e Quantification of the colocalization of mCherry-PIF5 foci with PHYB-CFP PBs in the experiments described in (d). Error bars represent the s.e. of at least three biological replicates. The source data underlying the hypocotyl measurements in (b), the immunoblots in (c), and the PB characterization in (e) are provided in the Source Data file.

Fig. 3. Increasing PHYB abundance provokes a biphasic PIF5 response.

a Immunoblots showing PHYB and PIF5 levels in 4-d-old phyB-9, Col-0, gPBC-25, gPBC-29 and PBC seedlings grown under 10 μmol m⁻² s⁻¹ R light at 21 ^oC. 4-d-old dark-grown Col-0 and R-light-grown pifq were positive and negative controls for PIF5, respectively. The immunoblot experiments were independently repeated three times with similar results. b Quantification of the PHYB and PIF5 levels shown in (a). Error bars represent the s.d. of three independent replicates. The centers of the error bars indicate the mean values. Different lowercase and uppercase letters denote statistically significant differences in the abundance of PHYB and PIF5, respectively (ANOVA, Tukey’s HSD, multiplicity adjusted p < 0.05, n = 3 biological replicates). c Quantitative real-time PCR (qRT-PCR) analysis of the transcript levels of PHYB and PIF5 in seedlings shown in (a). Different lowercase and uppercase letters denote statistically significant differences in PHYB and PIF5 transcripts, respectively (ANOVA, Tukey’s HSD, multiplicity adjusted p < 0.05, n = 3 biological replicates). d Immunoblots showing the degradation kinetics of PIF5 in Col-0, gPBC-25, and PBC. 4-d-old Col-0, gPBC-25, and PBC seedlings were incubated with cycloheximide and collected at the indicated time points. Total proteins were extracted and subjected to immunoblotting analysis using anti-PIF5 antibodies. For (a, d), actin was used as a loading control. The asterisk indicates a nonspecific band. The relative PHYB or PIF5 levels normalized to the corresponding actin levels are shown. e Quantification of the relative PIF5 protein levels shown in (d). Error bars represent the s.d. of three independent replicates. The source data underlying the immunoblots in (a, b, d, e), and the qRT-PCR analysis in (c) are provided in the Source Data file.

Fig. 4. PHYB recruits and stabilizes PIF5 in PBs.

a Images of 4-d-old PBC and mCherry-PIF5/PBC (#1 and #9) seedlings grown under 10 μmol m⁻² s⁻¹ R light at either 16 ^oC or 27 ^oC. b Hypocotyl length measurements of the seedings described in (a). Error bars represent the s.d., and the centers represent the mean (n ≥ 32 seedlings). Samples labeled with letters exhibited statistically significant differences in hypocotyl length (ANOVA, Tukey’s HSD, multiplicity adjusted p < 0.05). c Immunoblots showing the steady-state levels of PIF5 and mCherry-PIF5 in 4-d-old pif5-3, Col-0, PBC, and mCherry-PIF5/PBC (#1, #9) seedlings grown under 10 μmol m⁻² s⁻¹ R light at either 16 ^oC or 27 ^oC. d Confocal images showing the colocalization of mCherry-PIF5 with PHYB-CFP in PBs in cotyledon pavement cells from 4-d-old mCherry-PIF5/PBC #9 seedlings grown under 10 μmol m⁻² s⁻¹ R light at either 16 ^oC or 27 ^oC. Scale bars are equal to 2 µm. e Quantification of the colocalization of mCherry-PIF5 foci with PHYB-CFP PBs in the experiments described in (d). Error bars represent the s.e. (n = 9 biological replicates for the 16 ^oC samples, n = 7 biological replicates for the 27 ^oC samples), and the centers represent the mean. f Fluorescence microscope images showing the localization of PHYB-CFP and mCherry-PIF5 in hypocotyl epidermal cells from 4-d-old dark-grown mCherry-PIF5/PBC #9 seedings. The DIC image shows the nucleus. Scale bars are equal to 5 µm. g Immunoblots showing the degradation kinetics of Myc-tagged mCherry-PIF5 in mCherry-PIF5/pif5-3 and HA-tagged mCherry-PIF5 in mCherry-PIF5/PBC. 4-d-old seedlings of mCherry-PIF5/pif5-3 #8 and mCherry-PIF5/PBC #9 were incubated with cycloheximide and collected at the indicated time points. For (c, g), PIF5 and mCherry-PIF5 were detected using anti-PIF5 antibodies. Actin was used as a loading control. The asterisk indicates a nonspecific band. The relative levels of PIF5 and mCherry-PIF5 normalized to the corresponding actin levels are shown below each lane. h Quantification of the relative mCherry-PIF5 levels shown in (g). Error bars represent the s.d. of four independent replicates. The source data underlying the hypocotyl measurements in (b), the immunoblots in (c, g, h), and the quantification of the colocalization of mCherry-PIF5 and PHYB-CFP in e are provided in the Source Data file.

Fig. 5. Reducing PB size accelerates PIF5 degradation.

a Images of 4-d-old Col-0, PBC and mCherry-PIF5/PBC #9 seedlings grown under either 10 μmol m⁻² s⁻¹ or 0.5 μmol m⁻² s⁻¹ R light (R-10 and R-0.5, respectively). b Hypocotyl length measurement of the seedlings described in (a). Error bars represent the s.d., and the centers represent the mean (n = 90 seedlings). Samples labeled with different letters exhibited statistically significant differences in hypocotyl length (two-tailed Student’s t-test, p < 0.05). c Confocal images showing the PB patterns in cotyledon pavement epidermal cells from the mCherry-PIF5/PBC seedlings described in (a). Scale bars equal 2 µm. d Quantification of the sizes of the PBs described in (c). Error bars represent the s.d., and the center represents the mean (n = 53 PBs for the R-10 samples, n = 72 for the R-0.5 samples). Different letters denote statistically significant differences in PB size (two-tailed Student’s t-test, p < 0.05). e Quantification of the percentage PHYB-CFP PBs with detectable mCherry-PIF5 fluorescence in mCherry-PIF5/PBC seedlings grown under 0.5 μmol m⁻² s⁻¹ R light. The error bar represents the s.d., and the center represents the mean (n = 30). f Immunoblots of PHYB-CFP, endogenous PIF5 and mCherry-PIF5 in 4-d-old PBC and mCherry-PIF5/PBC #9 seedlings grown under either 10 μmol m⁻² s⁻¹ or 0.5 μmol m⁻² s⁻¹ R light. PHYB-CFP was detected using anti-PHYB antibodies, and mCherry-PIF5 and PIF5 were detected using anti-PIF5 antibodies. Actin was used as a control. The relative levels of PHYB-CFP, endogenous PIF5, and mCherry-PIF5, normalized to the corresponding actin levels, are shown under each lane. The asterisk indicates a nonspecific band. g Immunoblots showing the degradation kinetics of mCherry-PIF5 in 4-d-old mCherry-PIF5/PBC #9 seedlings grown under either 10 μmol m⁻² s⁻¹ or 0.5 μmol m⁻² s⁻¹ R light. Seedlings were incubated with cycloheximide and collected at the indicated time points. Total proteins were extracted and subjected to immunoblotting analysis using anti-PIF5 antibodies. Actin was used as a loading control. The relative mCherry-PIF5 levels normalized to the corresponding levels of actin are shown. h Results of FRAP experiments showing normalized fluorescence recovery plotted over time for PHYB-CFP in the mCherry-PIF5/PBC #9 seedlings described in (a). i Results of FRAP experiments showing normalized fluorescence recovery plotted over time for mCherry-PIF5 in the mCherry-PIF5/PBC #9 seedlings described in (a). For (h, i), values represent the mean, and error bars represent the s.e. of at least three biological replicates. Solid lines represent the exponential fit of the data. MF: mobile fraction. t_1/2 represents the half-time of fluorescence recovery. The source data underlying the hypocotyl measurements in (b), the PB characterization in (d, e), the immunoblots in (f, g), and the FRAP results in (h, i) are provided in the Source Data file.

Fig. 6. A PB-enabled counterbalancing model for titrating PHYB-mediated environmental responses.

a Schematic illustration of the function of PBs in segregating the opposing PHYB signaling actions of PIF5 degradation and stabilization. The condensation of PHYB creates a PIF5-stabilizing environment in PBs to counteract PIF5 degradation in the surrounding nucleoplasm. PHYB promotes PIF5 degradation in the nucleoplasm by triggering its phosphorylation and subsequent ubiquitylation by the CRL4^COP1/SPA E3 ubiquitin ligases^,,. PHYB serves as a scaffold component to recruit PIF5 as a client to PBs via direct interaction. PHYB stabilizes PIF5 by selectively recruiting COP1 and SPAs, but not the CRL4 core, to PBs^,,. The high concentration of PHYB in PBs disrupts the COP1-SPA complex via distinct interactions of COP1 with PHYB’s N-terminal photosensory module and SPAs with PHYB’s C-terminal output module^–,,. b Model for a PB-enabled counterbalancing mechanism to titrate nucleoplasmic PIF5 and environmental responses. Changes in the intensity and composition of light (and also in temperature) directly control the amount of active PHYB and thus the size of PBs to regulate the PB-to-nucleoplasm partitioning of PHYB, PIF5, and other PB constituents, thereby titrating the nucleoplasmic PIF5 and its signaling output. Strong R light increases the amount of active PHYB and enlarges PBs, thereby sequestering a greater fraction of PHYB and PIF5 in PBs, which stabilizes PIF5 and simultaneously reduces the nucleoplasmic PIF5 and its signaling output. This mechanism allows hypocotyl to grow slowly in the light. Dim R light or shade reduces the amount of active PHYB and PB size, thereby enhancing the nucleoplasmic fraction of PHYB and PIF5, simultaneously promoting PIF5 degradation and its transcriptional output. The latter mechanism accelerates hypocotyl elongation.